Studies of hair and skin continue to be at the forefront of regenerative medicine. Skin substitutes were among the earliest products to be developed using principles of tissue engineering, and the success of these ventures is evident in the clinical use of several commercially available products. In addition, hair restoration is one of the fastest growing areas of cosmetic therapies for both men and women.
The current clinical “gold standard” for treating major skin injuries involves the use of split-thickness skin autografts, which involves transplanting the epidermis with a portion of the dermis from one location on a patient to another. In cases where there is insufficient donor skin to cover the wounds, however, skin substitutes may be used. Skin substitutes available today have varied compositions, but generally comprise a nonliving collagen matrix and different combinations of keratinocytes and fibroblasts. For example, APLIGRAF® (Organogenesis, Inc., Canton, Mass.), which is reported to be the most clinically successful composite skin substitute currently available, is composed of allogeneic neonatal fibroblasts in bovine type I collagen overlaid with allogeneic neonatal keratinocytes.
However, currently available skin substitutes cannot perform all the functions of normal skin. For example, hair follicle (HF) neogenesis is not observed using any currently available skin substitute, which limits their use in patients. HFs and their associated sebaceous glands are important for appearance, skin hydration, barrier formation, and protection against pathogens. In addition, HFs store epidermal stem cells that may be called upon during wound healing. Thus, skin with HFs heals more rapidly than skin without HFs. In addition, any stem cells that might exist in skin lacking HFs are located in superficial layers of the epidermis, making the cells susceptible to loss through minor trauma and damage through ultraviolet light. Thus, treatments that involve neogenesis of normal HFs would find much wider application for restoring normal skin function and appearance.
During embryogenesis, mesenchymal cells signal the overlying epithelium to induce HF formation, and in adults a specialized group of mesenchymal cells, the dermal papilla (DP) cells, have been shown to retain the capacity to induce HF regeneration (Hardy 1992, Reddy et al., 2001, Gharzi et al., 2003). DP cells from rodents induce HFs in a variety of assays (reviewed in Ohyama et al., 2009), but it has been difficult to grow human DP cells that maintain inductive capacity in culture (Ohyama et al., 2013). This is a significant problem since DP cells must be enriched in culture to expand the cells needed for successful clinical use. Recent technological advances have enabled the use of human cells to form chimeric HFs, for example by combining human keratinocytes and rodent mesenchymal cells in chamber assays (Ehama et al., 2007), by combining human scalp dermal papilla cells and mouse epidermal keratinocytes in flap grafts (Qiao et al., 2009), or by injecting human DP cells, grown as spheroids, together with mouse epidermal cells in reconstitution or “patch” assays (Kang et al., 2012).
However, while chimeric HFs are highly valuable as investigative tools, they lack clinical utility because the HFs produced by these methods are not fully human constructs (but instead are chimeric rodent/human constructs), are not completely developed, contain hair shafts in the wrong anatomical location, do not exhibit long-term graft survival and normal HF cycling, and/or do not form HFs that contain sebaceous glands. In addition, HFs produced by such methods tend to grow in variable and uncontrollable directions, resulting in unnatural looking hair. Thus, the follicles produced by such methods are not useful for human HF neogenesis in skin lacking hair follicles.
Thus, a need exists for methods and compositions capable of generating morphologically-correct, fully-developed, non-immunogenic human hair follicles. Such methods and compositions would be useful for treating conditions such as full- or partial-thickness skin loss, wounds, burns, scars, and hair loss. The present invention fills these needs by providing cellular compositions capable of hair growth, neogenesis, and regeneration.
The present invention provides compositions in the form of skin substitutes and microspheres comprising neural crest-derived mesencymal cells, wherein the skin substitute is capable of inducing hair follicles that are morphologically-correct and are useful in any application requiring hair follicle formation/neogenesis, or in any condition where hair follicle formation/neogenesis is desired.
In one embodiment, the skin substitute or microsphere comprises neural crest-derived mesenchymal cells. In some embodiments, the skin substitute or microsphere further comprises epithelial cells, optionally with collagen. In another embodiment, the skin substitute or microsphere comprises scalp- or face-derived mesenchymal cells and epithelial cells, optionally with collagen. In another embodiment, the skin substitute or microsphere comprises epithelial cells and scalp- or face-derived mesenchymal cells, wherein the mesencymal cells are neural crest-derived, optionally with collagen. In yet another embodiment, the skin substitute or microsphere comprises epithelial cells and hair follicle dermal cells, optionally with collagen. In some aspects the epithelial cells are keratinocytes. In some aspects the skin substitute comprises cells of human origin only.
In various embodiments, a skin substitute is provided, comprising isolated neural crest-derived mesenchymal cells and/or epithelial cells. In some embodiments, the epithelial cells are keratinocytes, and/or the mesenchymal cells are hair follicle dermal cells (e.g., one or more of dermal papilla cells, dermal sheath cells, or hair follicle dermal cells derived from scalp or face). In some embodiments, the hair follicle dermal cells are derived from frontal, temporal, mid scalp, top of head, vertex, or parietal region of the scalp. In some embodiments, the hair follicle dermal cells are not derived from an occipital or nape region of the scalp. In various embodiments, the skin substitutes described above contain epithelial cells and neural crest-derived mesenchymal cells from a human. In some embodiments, the skin substitutes further comprise collagen. In some embodiments, the keratinocytes or keratinocyte-like cells are induced pluripotent stem (iPS) cells differentiated into keratinocytes or keratinocyte-like cells. In some embodiments, the mesenchymal cells and epithelial cells are taken from the same donor and/or the same body regions of a donor (e.g., the mesenchymal cells and epithelial cells are taken from tissue in the same donor region, e.g., keratinocytes and mesenchymal cells from the frontal scalp). In some embodiments, the mesenchymal cells and epithelial cells are taken from different donors and/or different body regions of a donor (e.g., the mesenchymal cells and epithelial cells are not taken from tissue in the same donor region, e.g., keratinocytes and mesenchymal cells from the frontal scalp).
In various embodiments, a skin substitute is provided, comprising epithelial cells and hair follicle dermal cells, wherein the hair follicle dermal cells are not derived from an occipital or nape region of the scalp. In some embodiments, the epithelial cells are keratinocytes, and/or the mesenchymal cells are hair follicle dermal cells (e.g., one or more of dermal papilla cells, dermal sheath cells, or hair follicle dermal cells derived from scalp or face). In some embodiments, the hair follicle dermal cells are derived from frontal, temporal, mid scalp, top of head, vertex, or parietal region of the scalp. In various embodiments, the skin substitutes described above contain epithelial cells and neural crest-derived mesenchymal cells from a human. In some embodiments, the skin substitutes further comprise collagen. In some embodiments, the skin substitutes are combined in therapeutically effective concentrations (e.g., concentrations not naturally found in combination in host tissue) and/or stored in a non-naturally occurring culture medium (e.g., Hanks media, keratinocyte-conditioned medium, or other cell culture media).
In various embodiments, the skin substitutes described above comprise mesenchymal cells (e.g., hair follicle dermal cells) provided within a matrix, e.g., a ground substance matrix or a collagen matrix such as a collagen type I matrix. In various embodiments, the skin substitutes described above are provided in a suspension such as a microsphere.
In various embodiments, the skin substitutes described above comprise keratinocytes that are from one or more of neonatal foreskin keratinocytes, adult keratinocytes, or keratinocyte-like cells derived from pluripotential stem cells or from epithelial cells. In some embodiments, the epithelial cells are primary cells or early passage cells (e.g., first through fourth passage, more preferably first or second passage). In some embodiments, the primary or early passage epithelial cells are mesenchymal cells. In some embodiments, the primary or early passage epithelial cells are hair follicle dermal cells. In some embodiments, the cells are the cells are passaged in keratinocyte-conditioned medium.
In various embodiments, the epithelial cells and mesenchymal cells are autologous. For instance, the epithelial cells and hair follicle dermal cells can be autologous. In some embodiments, the cells are allogenic.
In certain aspects of this invention, the neural-crest derived mesencymal cells, the scalp- or face-derived mesencymal cells, and the hair follicle dermal cells, including dermal papilla cells and dermal sheath cells, are not derived from an occipital or nape region of the scalp. For example, neural-crest derived mesencymal cells, the scalp- or face-derived mesencymal cells, and the hair follicle dermal cells, including dermal papilla cells and dermal sheath cells present in any composition or skin substitute described herein may be isolated from frontal, temporal, mid scalp, top of head, vertex, or parietal region of the scalp or from the face.
Also disclosed herein are methods for transplanting cells capable of inducing human hair follicles, comprising delivering to a human subject any one of the skin substitutes discussed above. In some embodiments, the epithelial cells discussed above are transplanted on their own (e.g., by coating, implanting, and/or injecting the cells into or onto a transplant site in a patient). In some embodiments, the mesenchymal cells discussed above are transplanted on their own (e.g., by coating, implanting, and/or injecting the cells into or onto a transplant site in a patient). In some embodiments, the epithelail cells are transplanted in combination with the mesenchymal cells. In some embodiments, the epithelail cells are transplanted in combination with the mesenchymal cells at therapeutically effective concentrations (e.g., concentrations not naturally found in combination in host tissue). In some embodiments, the transplanted cells can be used to induce hair follicle growth or hair follicle neogenesis.
Also disclosed are methods of treatment comprising transplanting the skin substitutes, as described above. In some embodiments, the subject to be treated with any of the compositions or methods described above has a partial-thickness skin loss, full-thickness skin loss, a wound, a burn, a scar, or hair loss. In some embodiments, transplanting the skin substitute induces eccrine glands and/or sebaceous glands.
In some embodiments, the epithelial cells and/or mesenchymal cells are for use in the methods described above, or are formulated for use in the methods, or are used in the preparation of a medicament for use in the methods described above.
Also disclosed herein are microspheres comprising neural crest-derived mesenchymal cells and/or epithelial cells. In some embodiments, the microspheres comprise both mesenchymal cells (e.g., hair follicle dermal cells) and epithelial cells (e.g., keratinocytes). In some embodiments, the mesenchymal cells are hair follicle dermal cells, such as dermal papilla cells, dermal sheath cells, or hair follicle dermal cells derived from scalp or face). In certain embodiments, the scalp or face-derived hair follicle dermal cells are from a frontal, temporal, mid scalp, top of head, vertex, or parietal region of the scalp, but not derived from an occipital or nape region of the scalp. In some embodiments, the the epithelial cells and neural crest-derived mesenchymal cells in the microspheres are human cells, and may further comprise collagen. For example, in certain embodiments, the microsphere comprises scalp- or face-derived mesenchymal cells (e.g., human cells), wherein the mesenchymal cells are not derived from an occipital or nape region of the scalp.
In some embodiments, the microspheres described above can further comprise a matrix, e.g., a ground substance matrix or a collagen matrix such as a collagen type I matrix to contain the dermal. In some embodiments, the keratinocytes in the microspheres are from one or more of neonatal foreskin keratinocytes, adult keratinocytes, or keratinocyte-like cells derived from pluripotential stem cells or from epithelial cells. In some embodiments, the epithelial cells in microspheres are primary cells or early passage cells (e.g., first through fourth passage, more preferably first or second passage). In some embodiments, the primary or early passage epithelial cells are mesenchymal cells. In some embodiments, the primary or early passage epithelial cells are hair follicle dermal cells. In some embodiments, the cells are the cells are passaged in keratinocyte-conditioned medium.
In various embodiments, the microspheres described above comprise autologous mesenchymal cells and/or epithelial cells. In some embodiments, the mesenchymal cells and/or epithelial cells are allogenic.
In various embodiments, methods are disclosed herein for transplanting cells capable of inducing human hair follicles to a subject, comprising delivering to a human subject any of the microspheres described above. In some embodiments, the microsphere is subdermally or intradermally delivered to a subject. In various embodiments, a method for inducing hair follicle growth or hair follicle neogenesis is provided, comprising delivering to a human subject any of the microspheres described above. In some embodiments, the microsphere is subdermally or intradermally delivered to a subject. In various embodiments, the subject has partial-thickness skin loss, full-thickness skin loss, a wound, a burn, a scar, or hair loss.
In various embodiments, a composition is provided herein for use in inducing hair follicle growth or hair follicle neogenesis in a subject, comprising any of the microspheres described above. Also disclosed herein are the microspheres described above for use in the manufacture of a medicament for inducing hair follicle formation or for inducing hair follicle neogenesis in a subject. In various embodiments, the subject has partial-thickness skin loss, full-thickness skin loss, a wound, a burn, a scar, or hair loss.
Also disclosed herein are methods for making a skin substitute, comprising (a) mixing a culture of primary or early-passage neural crest-derived mesenchymal cells with a matrix; and (b) overlaying a culture of primary or early-passage epithelial cells onto the mixture of (a). For example, the method can comprise (a) mixing a culture of scalp- or face-derived mesenchymal cells with a matrix; and (b) overlaying a culture of primary or early-passage epithelial cells onto the mixture of (a), wherein the mesenchymal cells are not derived from an occipital region of the scalp. As another example, the method can comprise (a) mixing a culture of hair follicle dermal cells with a matrix; and (b) overlaying a culture of primary or early-passage epithelial cells onto the mixture of (a), wherein the hair follicle dermal cells are not derived from an occipital region of the scalp. In various embodiments, the matrix is a collagen matrix (e.g., a collagen type I matrix). In various embodiments, the cells are cultured in keratinocyte-conditioned medium.
In certain embodiments, the compositions described and exemplified herein induce hair follicle formation when provided to human subjects, wherein the hair follicle that is formed is fully human and therefore does not elicit a host immune response.